Processing Methods to Concentrate

Bioactives in Soy Products

International Forum
Emerging Technologies in Food Processing
September 23-25, 2009
Urbana-Champaign, IL

Elvira Gonzalez de Mejia

Associate Professor
Department of Food Science and Human Nutrition,
University of Illinois at Urbana-Champaign
Department of Food Science and Human Nutrition
Agricultural, Consumer and Environmental
Sciences

http://www.aces.uiuc.edu
 OUTLINE
  Soybean components and their concentration through
processing

  Lunasin as a model

  Analysis and characterization of lunasin

  Process to isolate lunasin

  Optimization of lunasin concentration in soybean

  Mechanisms of action of lunasin and health benefits

  Conclusions

  Future Studies
 Objective

To offer an overview of the processing

methods to concentrate bioactives in soy
products. Lunasin as a model
Distribution in soybean world
production
Soybean production

  U.S. soybean production revenue was over 1 trillion

dollars in 2006 (3.188 billion bushels) (USDA, 2007).
 Bioactive components in
soybean

  Lower prevalence of high

plasma cholesterol, bowel
  Saponins   and kidney cancer,
  Bioac2ve  pep2des  
diabetes mellitus, and
  Isoflavones   obesity. Peptides are
potential bioactive
  Lunasin   components of soybean
(Messina et al., 2006).
  Bowman  Birk  inhibitor  
 Soybean
Composition

Phytochemicals

Soy    
Isoflavones  

Glycoside Aglycone
Genistin Genistein
Daidzin Daidzein
Glycitin Glycitein
 Saponins
Antiviral, antifungic,
diuretic,
antiinflammatory
(Berhow, 2006; Dia et al.,
2008)

Hipocolesterolemic
(Potter, 1995 ;
Lee et al., 2005)
Soy is a good source of bioactive peptides

α-conglycinin, β-subunit

Wang  and  de  Mejia.2005.  CRFSFS 4(4):63-­‐78  

 Soybean processing flow chart
Soybean
Hulls Oil
Cleaning Fat
Soybeans Flaking
Cracking Soy Chips Extraction
Edible
Defatted
Grinding Flakes
Soy
Grinding and Sizing
Grits
Sugar Protein
Water Removal Extraction
Soy Flour
Blending Drying Protein
Precipitates
Texturing Soy
Protein Drying
Drying Concentrate
Screening (SPC) Soy
Protein
TVP Isolate (SPI)
Products
Commercial soybean products
Food processing affects bioactive components

–  Heat
•  Heating of soybean  Remove trypsin inhibitor (Brandon
et al., 1991)

•  Heating of soymilk  Unfolding, dissociation,

denaturation, and aggregation of proteins (Kwok and Niranjan,
1995)

–  Fermentation
•  Soy proteins  Smaller peptides, amino acids, and
ammonia (Whitaker, 1978; Shreffler et al., 2001)
Value-­‐added  soy  products  
 Soybean Lunasin
Predicted secondary structure
of lunasin

Amino acid sequence of

lunasin

de  Lumen,  B.  O.,  Nutr  Rev,  63,  2005      

Wang  et  al.,  J  AOAC  Intl,  91,  2008    
 Lunasin Facts
 Activity is found in processed legumes and
dry cereals. It is heat stable, surviving
temperatures up to 100°C for 10 min.
 Resistant to proteolytic digestion, gets
absorbed, and enters target tissues.
 Major component of the Bowman-Birk
protease inhibitor, a cancer preventive
component from soybeans.
Effect of lunasin on skin tumorigenesis in
female Sencar mice

Galvez et al., Cancer Research 61: 7473-7478, 2001

 Isolation and Purification of Lunasin
Anion exchange
Defatted soybean flour
chromatography
In water (1:5, w/v)
DEAE, pH 7.5
Ultrafiltration
3 kDa
Size exclusion
SDS-PAGE, Western blot, chromatography
ELISA, 25 kDa
RP-HPLC,
MALDI–TOF,
LC-MS-MS
Size exclusion
chromatography Lunasin
7 kDa
(86%)
Enzyme Linked Immuno Sorbent Assay
 Factors affecting purification of lunasin

pH 8.2
Soy flour (Arcon F from ADM), material/solvent ratio (1:5) suspended Effect of pH on binding of soy protein and synthetic
in different solvents and extracted at 40 °C for 70 min with sonication lunasin on DEAE resin

Wang et al., 2008. Analysis of soybean protein-derived peptides and the effect of
cultivar, environmental conditions, and processing on lunasin concentration in
soybean and soy products. JAOAC Int. 91 (4): 936-946.
 Factors affecting purification of lunasin

Effect of elution salt concentration on recovery of Purification of lunasin from soy proteins using
soy protein and synthetic lunasin from DEAE resin ion exchange chromatography, XK 50/30 column
packed with DEAE resin

Wang et al., 2008. JAOAC Int. 91 (4): 936-946.

 DEAE fractions show similar chromatographic
peaks than synthetic lunasin in RP-HPLC using
Vydac C4 column
B

Wang et al., 2008. JAOAC Int. 91 (4): 936-946.

Factors affecting purification of lunasin

14
11
6

16

Lunasin purification with gel filtration Lunasin concentration of size exclusion

chromatography Superdex 75 (XK 26/70) and eluted fractions using 25 kDa molecular weight
with 20 mM Tris-HCl, pH 7.5 containing 0.15 M NaCl cut-off
at 4 ml/min

Wang et al., 2008. JAOAC Int. 91 (4): 936-946.

5 kDa

Gonzalez de Mejia and Dia. Lunasin and lunasin-like peptides

inhibit inflammation through suppression of NF-kB pathway in
the macrophage. Peptides, 2009. doi:10.1016/j.peptides.
2009.08.005
Dose response cytotoxicity of lunasin
enriched flour on L1210 cells

IC50 = 0.34 mg/ml

P < 0.05
Cell viability (%)

Lunasin enriched flour (mg/ml)

Wang  et  al.,  JAFC,  2008  

Cytotoxicity of lunasin and Bowman Birk
inhibitor on L1210 cells

Lunasin  (98%  purity)  and  BBI  (95%  purity)  inhibit  prolifera>on  

of  leukemia  cells  in  a  dose-­‐dependent  manner  
Wang  et  al.,  JAFC,  2008  
Lunasin induces apoptosis of leukemia cells

Untreated

Treated
1 mg LEF/mL 24 h

50 µM
Hoechst  stain                  Caspase  stain  
Gonzalez de Mejia, et al., Lunasin, with an arginine-glycine-aspartic acid motif, causes
apoptosis to L1210 leukemia cells by activation of caspase-3. Mol. Nutr. Food Res. 2009.
In Press. mnfr.200900073
Lunasin induces apoptosis of leukemia cells
1: Control 2: 5 µM. 3: 15 µM. 4: 50 µM. 5: 250 µM lunasin

4% 3.5% 7.2% 23.2% 43.2%

Gonzalez de Mejia, et al., Lunasin, with an arginine-glycine-aspartic acid motif, causes

apoptosis to L1210 leukemia cells by activation of caspase-3. Mol. Nutr. Food Res. 2009.
In Press. mnfr.200900073
 Comparison of the anti-inflammatory properties of
the lunasin-like peptides and their mixture IC30 (µM)

Peptide COX-2 PGE2 iNOS NO

5 kDa 13.0a 17.5a 15.7a 11.8b

8 kDa 33.9b 38.8b 32.7b 32.9c

14 kDa 15.1a 33.6b 33.6b 23.6c

Mixture 114.8c 223.9c 56.3c 1.8a

Gonzalez de Mejia and Dia. Lunasin and lunasin-like peptides

inhibit inflammation through suppression of NF-kB pathway in
the macrophage. Peptides, 2009. doi:10.1016/j.peptides.
2009.08.005
Industrial Samples

–  Samples from different parts of the soybean

process, including:
–  Soy Flour - Soy Powder

–  Soy Protein Concentrate - Hydrolyzed Soy

Protein
–  Soy Protein Isolate - Isoflavone
Concentrate
–  Soy Flakes - Saponin
Concentrate

–  Samples from selected industrial waste effluents of

the soy protein process studied for the isolation and
purification of lunasin.
Lunasin concentration among different
soy products
Lunasin in Chinese germplasm

Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.

Lunasin content in commercially available
isoflavone products (mg/g solid material)

Now Extra Strength Soy Isoflavones, 60 mg 1.0

(soy isoflavone extract, rice flour)

Vitamin World Soy Isoflavones 23 mg (soy 1.4

extract)

PhytoNutramins Isoflavones 11 mg 3.1

(isoflavones from soybean flour)

TwinLab Soy Germ Isoflavone Caps 10 mg 4.8

Soy Care for Menopause 25 mg (soy extract, 11.6
soy protein concentrate)

Low Isoflavone 15.1

Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.
Lunasin in waste streams

Current research on methods to purify

lunasin from soy molasses

Gonzalez de Mejia, et al. J Agric. Food Chem., 2004.

Germination as a process to improve
bioactive compounds
•  Increase nutritive
value
•  Reduce
antinutritional factors
•  Increases methionine
value
(Barcelos et al., 2002).
 Experimental design to obtain
germinated soy flour
Levels Soybean
Independent
Variables
-α -1 0 +1 +α Sanitation
Grinding
x 12, 21, 42, 63, 72 h
Time (h) 12 21 42 63 72
1
Germination 18, 20, 25, 30, 32 ºC

Drying
x Temperature
18 20 25 30 32 Homogenization
2 (ºC)

±⏐α⏐=1,41
Integral germinated soy flour
Paucar-Menacho, et al. Optimization of germination time and temperature on the concentration of bioactive
compounds in Brazilian soybean cultivar BRS 133 using response surface methodology. Food Chem. 118 (3), 2010.
10.1016/j.foodchem. 2009.07.011.
 Optimization of lunasin
concentration by germination

Paucar-Menacho, et al. Effect of time and temperature on bioactive compounds of

germinated Brazilian soybean cultivar BRS 258. Food Res. Int. 2009. http://
dx.doi.org/10.1016/j.foodres.2009.09.016
 Optimization of lunasin
concentration by germination

High concentration lunasin

and low concentration of
lectin and lipoxygenase

High concentration
isoflavones aglycones and
saponins

Paucar-Menacho, et al. A high-protein soybean cultivar contains lower

isoflavones and saponins but higher minerals and bioactive peptides than a
low-protein cultivar. Food Chem., , 2009. 10.1016/j.foodchem.2009.09.062.
 Lunasin
Complete Model : R2 = 0,95
Ajusted Model : R2 = 0,91
Lunasin (mg/g PS) = 21,08 – 2,45x12 – 4,38 x22 – 4,03 x1x2

Paucar-Menacho, et al. Optimization of germination time and temperature on the

concentration of bioactive compounds in Brazilian soybean cultivar BRS 133 using response
surface methodology. Food Chem. 118 (3), 2010. 10.1016/j.foodchem. 2009.07.011.
 Lunasin bioavailability
 High-protein soybean isolated powder (90%
protein)
 Whole soybean chili dish prepared at the
National Research Soybean Laboratory (University
of Illinois)

Dia  et  al.  J.  Agric  Food  Chem.  (2009)  

 In  vitro  diges2on  experiment  

 SDS-­‐PAGE  
 Degree  of  hydrolysis  

Time (min)   0 10 20 30 45 60 90 120 150 180 190 200 210 220 235 250 280 310 340 370

pH 2   pH 7.5  
Pepsin   Pancrea2n  
Gonzalez de Mejia, et al., Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210
leukemia cells by activation of caspase-3. Mol. Nutr. Food Res. 2009. In Press. mnfr.200900073
Lunasin during simulated gastro-
intestinal digestion

Pepsin   Pancrea2n  

n=6, P< 0.05

Gonzalez de Mejia, et al., Lunasin, with an arginine-glycine-aspartic acid motif, causes apoptosis to L1210
leukemia cells by activation of caspase-3. Mol. Nutr. Food Res. 2009. In Press. mnfr.200900073
 Quantification of lunasin in blood of
men fed soy protein
Pre-loading of beads

Equilibration of beads

Adsorption of proteins/peptides (~ 50 µg/0.5 mg beads)

Desorption (Using different concentrations of NaCl)

Determination of lunasin in eluate: ELISA, MALDI-TOF,

LC/MS-MS

Dia  et  al.  J.  Agric  Food  Chem.  (2009)  

Lunasin in plasma of men after
soy protein consumption

Dia  et  al.  J.  Agric  Food  Chem.  57:  1260  (2009)  
Commercial peptide products
 Manufacturing of protein
hydrolysates/bioactive peptides
•   Batch  versus  con2nuous  process  

•   Hydrolysis  with/without  preserva2ves  

•   Hydrolysis  by:  
         Acid,  Alkali,  versus  Enzymes  and  Fermenta2on  Processes  

•   Downstream  Processing:  
           Filter  press  versus  Membrane  separa2ons  
           Centrifuge  versus  con2nuous  centrifuge  
           Chromatography,  Ion  exchange  resin  columns  
 Manufacturing of protein
hydrolysates/bioactive peptides
•   Downstream  processing:  

•   Pasteuriza2on:    
         Keale  hea2ng  versus  Plate  &  Frame  Pasteurizer  and  UHT    

•   Evapora2on:  
       Hea2ng  under  vacuum  versus  mul2ple  effect  falling  film  

•   Drying:  
     Drum  drying  versus  spray  drying  and  fluid  bed  drying  for  
     agglomerated  products.  
 Conclusions
•  Lunasin is present in most commercial soy
products in proportion to the amount of
protein (range 13 to 44 mg/g dry product)

•  Soy processing waste effluents contain

10.4 mg lunasin/g solid material
 Conclusions
Lunasin can be found in human circulation,
a key requirement for its biological
activity.
The average concentration of lunasin 1 h after
ingestion was 71.0 ng/mL equivalent to 0.21
mg of lunasin in a person containing 3 L of
plasma. This represents an average of
4.5% absorption (range of 2.2-7.8%).
 Conclusions
•  Lunasin and lunasin-like peptides
inhibited inflammation through
suppression of NF-κB activation

•  Lunasin has potential as a unique and

novel agent for human health and
wellness.
 The consumption of 25 grams per day of soy
protein recommended by the FDA for
reducing coronary heart disease risk supplies
250 mg of lunasin. Whether this is
physiologically relevant remains to be
determined in clinical trials.
  Identification of new bioactive soy
peptides and their in vivo
biological functions
  New commercial production
techniques
  Effect of industrial processing on
bioactive peptide generation
THANK  YOU  FOR  YOUR  ATTENTION    
Thank you!
 Thank you

USDA  Future  Foods  Ini2a2ve  

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